Genetic engineering of plants through manipulation of lignin biosynthesis

ABSTRACT

The invention pertains to the genetically down regulating a lignin pathway p-coumarate Co-enzyme A ligase (CCL) in trees.

FIELD OF THE INVENTION

[0001] The invention relates to genetically modifying trees throughmanipulation of the lignin biosynthesis pathway, and more particularly,to genetically modifying trees through the down regulation ofp-coumarate Co-enzyme A ligase (CCL) to achieve faster growth, and/oraltered lignin content, and/or altered lignin structure, and/or alteredcellulose content and/or disease resistance of the trees and to the useof promoters of the CCL genes to drive gene expression specifically inxylem tissue or specifically in epidermal tissues.

BACKGROUND OF THE INVENTION

[0002] Genetic engineering of forest tree species to conform to desiredtraits has shifted the emphasis in forest tree improvement away from thetraditional breeding programs during the past decade. Although researchon genetic engineering of forest trees has been vigorous, the progresshas been slow due.

[0003] The ability to make trees grow faster and be disease resistant toproduce the highest volume of wood in the shortest period of time hasbeen and continues to be the top objective of many forest productscompany worldwide. The ability to genetically increase the optimalgrowth of trees would be a commercially significant improvement. Fastergrowing trees could be used by all sectors of the forest and woodproducts industry worldwide.

[0004] Lignin, a complex phenolic polymer, is a major component in cellwalls of secondary xylem. In general, lignin constitutes 25% of the dryweight of the wood, making it the second most abundant organic compoundon earth after cellulose. Although lignin plays an important role inplants, it usually represents an obstacle to utilizing biomass inseveral applications. For example, in woodpulp production, lignin has tobe removed through expensive and polluting processes in order to recovercellulose.

[0005] Thus, it is desirable to genetically engineer plants with reducedlignin content and/or altered lignin composition that can be utilizedmore efficiently. Trees that could be genetically engineered with areduced amount of lignin would be commercially valuable. Thesegenetically engineered trees would be less expensive to pulp because, inessence, part of the pulping has already been performed due to thereduced amount of lignin.

[0006] Trees with increased cellulose content would also be commerciallyvaluable to the pulp and paper industry.

[0007] Disease resistance in plants is also a most desirable planttrait. The impact of disease resistance in trees on the economy offorest products industry worldwide is significant.

[0008] Promoters that target specific plant tissue could be useful inmanipulating gene expression to enable the engineering of traits ofinterest in specific tissue of plants, such as, xylem and epidermaltissues.

[0009] Although studies have revealed several general properties ofplant p-coumarate Co-enzyme A ligase (CCL), the role of CCL inregulating the synthesis of monolignols in response to different statesof development and environmental stress in tree species remains largelyunknown. Furthermore, multiple CCL isoforms are normally present inplants, channeling phenolic compounds to the biosynthesis of not onlylignin but also other phenylpropanoids, such as flavonoids. Since CCLisoforms have not been previously cloned from tree species for theidentification of their biochemical functions, the presence of CCLisoforms remains so far as a challenge to a specific control ofmetabolic flux to the lignin biosynthesis in tree species.

SUMMARY OF THE INVENTION

[0010] The invention provides a method to genetically alter treesthrough the down regulation of p-coumarate Co-enzyme A ligase (CCL).Such down regulation of CCL results in faster growth, and/or reducedlignin content, and/or altered lignin structure, and/or alteredcellulose content and/or disease resistance. The invention also providesfor genetically engineered trees which have been altered to downregulate p-coumarate Co-enzyme A ligase (CCL) to achieve faster growth,and/or reduced lignin content, and/or altered lignin structure, and/orincreased cellulose content and/or increased disease resistance. Theinvention also provides tissue specific promoters of the CCL genes thatcan be used to manipulate gene expression in target tissue such as xylemand epidermal tissues.

[0011] It is one object of the present invention to down regulatep-coumarate Co-enzyme A ligase (CCL) in trees.

[0012] It is another object of the present invention to provide a methodto genetically alter trees to grow faster.

[0013] It is another object of the present invention to provide a methodto genetically alter the growth of trees through manipulation the ligninpathway p-coumarate Co-enzyme A ligase.

[0014] It is another object of the present invention to providegenetically altered trees with an accelerated growth characteristic.

[0015] It is another object of the present invention to providetransgenic trees with an accelerated growth characteristic which havebeen genetically altered by down regulating lignin pathway p-coumarateCo-enzyme A ligase.

[0016] It is another object of the present invention to provide a methodto genetically alter trees to reduce their lignin content.

[0017] It is another object of the present invention to provide a methodto genetically alter the lignin content of trees through manipulation ofa lignin pathway enzyme.

[0018] It is another object of the present invention to geneticallyengineer trees which have reduced lignin content through manipulation oflignin pathway p-coumarate Co-enzyme A ligase.

[0019] It is another object of the present invention to providegenetically altered trees with a reduced lignin content.

[0020] It is another object of the present invention to providetransgenic trees with reduced lignin content which have been geneticallyaltered by down regulating the p-coumarate Co-enzyme A ligase (CCL).

[0021] It is another object of the present invention to provide a methodto genetically alter trees to change their lignin structure throughmanipulation of lignin pathway p-coumarate Co-enzyme A ligase.

[0022] It is another object of the present invention to provide treeswith altered lignin structure.

[0023] It is another object of the present invention to provide a methodto increase the cellulose content in trees.

[0024] It is another object of the present invention to provide a methodto increase the cellulose content of trees through the manipulation of alignin pathway enzyme.

[0025] It is another object of the present invention to provide treeswith increased cellulose content.

[0026] It is another object of the present invention to providetransgenic trees having increased cellulose content from the downregulation of CCL.

[0027] It is another object of the present invention to provide a methodto genetically alter trees to increase their disease resistance.

[0028] It is another object of the present invention to provide a methodto genetically alter trees to be more disease resistant throughmanipulation of the lignin pathway p-coumarate Co-enzyme A ligase.

[0029] It is another object of the present invention to geneticallyalter trees to increase their disease resistance to fungal pathogens.

[0030] It is another object of the present invention to provide treeswith increased disease resistance.

[0031] It is another object of the present invention to providetransgenic trees with increased disease resistance through downregulation of the lignin pathway p-coumarate Co-enzyme A ligase.

[0032] It is another object of the present invention to provide a methodusing a promoter of a CCL gene to target gene expression in specificplant tissue.

[0033] It is another object of the present invention to provide a methodusing a promoter of a CCL gene to target gene expression specifically inplant xylem.

[0034] It is another object of the present invention to provide a methodusing a promoter of the CCL gene to target gene expression specificallyin the epidermal tissues of plants.

[0035] It is another object of the present invention to provide a CCLgene promoter that targets gene expression specifically in the xylem ofplants.

[0036] It is another object of the present invention to provide a CCLgene promoter that targets gene expression specifically in the epidermaltissues of plants.

[0037] Other features and advantages of the invention will becomeapparent to those of ordinary skill in the art upon review of thefollowing drawing, detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 is a schematic of a phenylpropanoid pathway;

[0039]FIG. 2 is a diagram of Agrobacterium T-DNA construct pACCL1;

[0040]FIG. 3 is a restriction map of genomic clone PtCCL1g-4;

[0041]FIG. 4 is a restriction map of genomic clone PtCCL2g-11;

[0042]FIG. 5 is a restriction map of subcloned PtCCL1 gene promoterp7Z-4XS;

[0043]FIG. 6 is a restriction map of subcloned PtCCL2 gene promoterpSK-11HE

[0044]FIG. 7 is an Agrobacterium T-DNA construct of PtCCL1 promoter andGUS fusion gene, PtCCL1p-GUS; and

[0045]FIG. 8 is an Agrobacterium T-DNA construct of PtCCL2 promoter andGUS fusion gene, PtCCL2p-GUS.

[0046] Before one embodiment of the invention is explained in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description of the preferredembodiment. The invention is capable of other embodiments and of beingpracticed or being carried out in various ways. Also, it is to beunderstood that the phraseology and terminology used herein is for thepurpose of description and should not be regarded as limiting.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0047] The invention pertains to genetically down regulating a ligninpathway p-coumarate Co-enzyme A ligase (CCL). Trees which have beengenetically transformed to down regulate CCL will hereafter be calledtransgenic trees. Such down regulation can result in faster growingtrees. Such down regulation can result in a reduction in the lignincontent of the trees and/or altered lignin structure. Such downregulation can result in increased cellulose content. Such downregulation can result in increased tree disease resistance. Further, byusing a specific promoter of CCL, targeted tissue gene expression can beachieved in either the xylem or the epidermal tissues of the plant.

[0048] A. CCL

[0049] Lignin is synthesized by the oxidative coupling of threemonolignols (coumaryl, coniferyl and sinapyl alcohols) formed via thephenylpropanoid pathway as shown in FIG. 1. Reactions in thephenylpropanoid pathway include the deamination of phenylalanine tocinnamic acid followed by hydroxylations, methylations and activation ofsubstituted cinnamic acids to coenzyme A (CoA) esters. The CoA estersare then reduced to form monolignols which are secreted from cells toform lignin.

[0050] The products of the phenylpropanoid pathway are not only requiredfor the synthesis of lignin but also required for the synthesis of awide range of aromatic compounds including flavonoids, phytoalexins,stilbenes and suberin.

[0051] In angiosperms (hardwoods), lignin is composed of both coniferyland sinapyl alcohol and is classified as guaiacyl-syringyl lignin.Grasses synthesize a third precursor (p-coumaryl alcohol) which ispolymerized along with coniferyl and sinapyl alcohol. In gymnosperms(softwoods), lignin is composed of mainly coniferyl alcohol and isclassified as guaiacyl lignin.

[0052] In the phenylpropanoid pathway, CCL activates a number ofcinnamic acid derivatives, including p-courmaric acid, caffeic acid,ferulic acid, 5-hydroxyferulic acid and sinapic acid. The resultingproducts, CoA esters, serve as substrates for entry into various branchpathways, such as lignin, flavonoids, phytoalexins, stilbenes andsuberin. The esterification reactions catalyzed by CCL require highenergy and the reactions are not likely to occur without CCL. CCL isimportant in making a continuous flow of the lignin biosynthesispathway. CCL is also important because it is located at the branchingpoints of the phenylpropanoid metabolism. CCL is suggested to play apivotal role in regulating carbon flow into specific branch pathways ofthe phenylpropanoid metabolism in response to stages of development andenvironmental stress.

[0053] The basic properties of CCL are quite uniform. CCL depends on ATPas a cosubstrate and requires Mg²⁺ as a cofactor. The optimal pH for CCLranges from pH 7.0 to 8.5 and the molecular weight of CCL isoforms fromvarious plant species ranges from 40 kd to 75 kd. Most CCLs have highaffinity with substituted cinnamic acids. CCL has the highest activitywith p-coumaric acid.

[0054] CCL cDNA sequences have been reported for parsley, potato,soybean, loblolly pine, Arabidopsis, Lithosperum and tobacco. CCL geneshave been isolated and sequenced for parsley, rice, potato and loblollypine. The analysis of CCL cDNAs and genes indicates that CCL is encodedby multiple/divergent genes in rice, soybean, and Lithosperum, verysimilar genes in parsley, potato, tobacco and loblolly pine, and asingle gene in Arabidopsis. CCL promoters have been isolated andsequenced for parsley, rice and potato.

[0055] Alignment of deduced amino acid sequences of cloned plant CCLsequences reveals two highly conserved regions. The first conservedregion (SSGTTGLPKGV), proposed to designate a putative AMP-bindingregion, is very rich in Gly, Ser and Thr and is followed by a conservedLys. The second conserved region (GEICIRG) contains one common Cysresidue. The amino acid sequences of CCL from plants contain a total offive conserved Cys residues.

[0056] The CCL genes of parsley, potato and rice contain five exons andfour introns. The CCL genes also share the same exon/intron splicejunction sites but have different lengths of introns. The genomicsequences of loblolly pine CCL are composed of four exons and threeintrons. It has been found that two similar CCL genes of the samespecies may differ slightly in length of intron as shown in two parsleygenes (PC4CL1 and PC4CL2) and in two loblolly pine genes (LP4CL1 andLP4CL2).

[0057] By Northern blot analysis, it has been shown that CCL isexpressed in leaf, shoot tip, stem, root, flower and cell culture. Twosimilar CCL cDNAs in parsley, potato and tobacco have been shown to beexpressed at similar level in response to the environmental stress andduring different developmental stages. Two distinct CCL cDNAs in soybeanand Lithosperum have shown different expression levels when pathogens orchemicals were applied to the cell cultures. It appears that theexpression of the CCL genes is developmentally regulated and inducibleby many environmental stresses at the transcription level.

[0058] Genetic transformation with a CCL sequence can result in severalsignificant affects. The description of the invention hereafter refersto aspen, and in particular quaking aspen (Populus tremuloides Michx)when necessary for the sake of example. However, it should be noted thatthe invention is not limited to genetic transformation of aspen. Themethod of the present invention is capable of being practiced for othertrees, including for example, other angiosperms, other gymnosperm foresttree species, etc.

[0059] Preferably, the CCL down regulation is accomplished throughtransformation with a homologous CCL sequence in an antisenseorientation. However, it should be noted that a heterologous antisenseCCL sequence could be utilized and incorporated into a tree species todown regulate CCL if the heterologous CCL gene sequence has a highnucleotide sequence homology, approximately higher than 70%, to theendogenous CCL gene sequence of that tree species.

[0060] In addition, trees transformed with a sense CCL sequence couldalso cause a sequence homology-based cosuppression of the expression ofthe transgene and endogenous CCL gene, thereby achieving down regulationof CCL in these trees.

[0061] B. Isolation of CCL cDNAs

[0062] The present invention utilizes a homologous CCL sequence togenetically alter trees. The preferred embodiment of the invention asfurther described below utilizes a cDNA clone of the quaking aspen CCLgene.

[0063] Two aspen (Populus tremuloides Michx) cDNAs encoding two distinctCCL isoforms, PtCCL1 and PtCCL2 have been cloned. PtCCL1 cDNA is ligninpathway-specific and is different from PtCCL2 cDNA, which is involved inflavonoid synthesis. The cloning of PtCCL1 and PtCCL2 cDNAs and theidentification of their biochemical functions will be discussed in morelength below. PtCCL1 and PtCCL2 genomic closes including their 5′-endregulatory promoter sequences were also isolated. The promoter of PtCCL1(PtCCL1p) directs xylem tissue-specific gene expression in a plant,whereas the promoter of PtCCL2 (PtCCL2p) drives the expression of genesspecifically in epidermal tissues of stem and leaf of a plant. Thesetissue specific promoters will be discussed in more length in Section Ibelow.

[0064] Two CCL cDNAs, PtCCL1 and PtCCL2, have been isolated from quakingaspen using either a conventional cDNA library screening method or aPCR-based cDNA cloning method. It should be noted that the methodsdescribed below are set forth as an example and should not be consideredlimiting. These CCL cDNA clones are available from MichiganTechnological University, Institute of Wood Research, Houghton, Mich.

[0065] Young leaves and shoot tips are collected from greenhouse-grownquaking aspen (Populus tremuloides Michx). Differentiating xylem andsclerenchyma are collected from three to four year old quaking aspen.The bark is peeled from the tree exposing the developing secondary xylemon the woody stem and the sclerenchyma on the inner side of the bark.Developing secondary xylem and sclerenchyma are scraped from the stemand bark with a razor blade and immediately frozen in liquid nitrogenuntil use.

[0066] Total RNA is isolated from the young leaves, shoot tips, xylemand sclerenchyma following the method of Bugos RC et al. (1995), RNAIsolation from Plant Tissue Recalcitrant to Extraction in Guanidine,Biotechniques 19(5):734-737. Poly(A)⁺RNA is purified from total RNAusing Poly(A)⁺mRNA Isolation Kit from Tel-test B, Inc. A unidirectionalLambda gt22 expression cDNA library was constructed from the xylem mRNAusing Superscript S System from Life Technologies, Inc. and GigapackPackaging Extracts from Stratagene. The PtCCL1 cDNA was obtained byscreening the cDNA library with a ³²P-labeled parsley 4CL cDNA probe.The parsley 4CL cDNA (pc4CL2) has Genbank accession number X13325.

[0067] The PtCCL2 cDNA was obtained by RT-PCR. The reverse transcriptionof total RNA isolated from shoot tips was carried our using theSuperscript II reverse transcriptase from Life Technologies. Two senseprimers (R1S, 5′-TTGGATCCGGIACIACIGGIYTICCIAARGG-3′ and H1S,5′-TTGGATCCGTIGCICARCARGTIGAYGG-3′) are designed around the firstconsensus AMP-binding region of CCL that was previously discussed. Oneantisense primer (R2A, 5′-ATGTCGACCICGDATRCADATYTCICC-3′) is designedbased on the sequence of the putative catalytic motif GEICIRG. One fifthof the reverse transcription reaction (4 Tl) is used as the template ina 50 Tl PCR reaction containing 1×reaction buffer, 200 TM eachdeoxyribonucleotide triphosphate, 2 TM each R1S and oligo-dT (20 mer)primers, and 2.5 units of Taq DNA polymerase. The PCR reaction mixturewas denatured at 940 C for 5 minutes followed by 30 cycles of 940 C/45seconds, 500 C/1 minute, 720 C/90 seconds and is ended with a 5 minuteextension at 720 C. 2 μl of the PCR amplification products are used fora second run PCR re-amplification using primers H1S and R2A. A 0.6 kbPCR fragment is cloned using the TA Cloning Kit from Invitrogen and usedas a probe to screen an aspen genomic library to obtain the PtCCL2genomic clone. Two primers (2A, 5′-TCTGTCTAGATGATGTCGTGGCCACGG-3′ and2B, 5′-TTAGATCTCTAGGACATGGTGGTGGC-3′) are designed based on the genomicsequence of PtCCL2 at around the deduced transcription start site andstop codon for the cloning of PtCCL2 cDNA by RT-PCR as described aboveusing total RNA isolated from shoot tips.

[0068] The DNA sequences of PtCCL1 and PtCCL2 cDNA were determinedusing—Taq Cycle Sequencing Kit from Amersham.

[0069] The PtCCL1 cDNA has an open reading frame of 1620 bp whichencodes a polypeptide of 540 amino acid residues with a predictedmolecular weight of 59 kd and pI of 5.8. The nucleotide sequence of theaspen CCL cDNA clone PtCCL1 is set forth as SEQ ID NO:1. The deducedamino acid sequence for the aspen CCL1 protein is set forth as SEQ IDNO:2.

[0070] The PtCCL2 cDNA has an open reading frame of 1713 bp whichencodes a polypeptide of 571 amino acid residues with a predictedmolecular weight of 61.8 kd and pI of 5.1. The nucleotide sequence ofthe aspen CCL cDNA clone PtCCL2 is set forth as SEQ ID NO:3. The deducedamino acid sequence for the aspen CCL2 protein is set forth as SEQ IDNO:4.

[0071] The aspen PtCCL1 cDNA shares a 59-74% identity at the nucleotidelevel and 59-81% identity at the amino acid level with other priorreported CCL cDNAs and genes, whereas the PtCCL2 cDNA shares a 60-73%identity at the nucleotide level and 57-74% at the amino acid level withother CCL cDNAs and genes as set forth in the following table. TABLE 1Comparison of PtCCL1 and PtCCL2 Nucleotide and Predicted Amino AcidSequence to Each Other and Other CCL Sequences DNA AMINO IDEN- DNA AMINOACID ACID TITY % IDENTITY % IDENTITY % IDENTITY cDNA* PtCCL1 PtCCL2PtCCL1 % PtCCL2 PtCCL1 62 63 LE4CL1 69 62 71 64 LE4CL2 60 71 59 73 GM1474 67 81 69 GM16 62 73 65 73 NT4CL1 67 62 75 74 NT4CL2 66 63 75 66PC4CL1 66 64 71 64 PC4CL2 66 63 72 64 ST4CL1 67 63 75 64 AT4CL 66 63 7061 LP4CL 61 64 63 67 OS4CL1 59 60 59 57

[0072] The results of sequence analysis, phylogenetic tree and genomicSouthern blot analysis indicate that PtCCL1 and PtCCL2 cDNAs encode twodistinct CCLs that belong to two divergent gene families in aspen. Thededuced amino acid sequence for the PtCCL2 protein contains a longerN-terminal sequence than the PtCCL1 protein but shows profoundsimilarity in the central and C-terminal portions of protein to thePtCCL1 protein.

[0073] PtCCL1 and PtCCL2 cDNAs display distinct tissue-specificexpression patterns. The PtCCL1 sequence is expressed highly in thesecondary developing xylem and in the 6th to 10th internodes whereas thePtCCL2 sequence is expressed in the shoot tip and leaves. Thesetissue-specific expression patterns were investigated by fusingpromoters of PtCCL1 and PtCCL2 genes to a GUS reporter gene. The tissuespecific promoters for PtCCL1 and PtCCL2 will be discussed in morelength in Section I below.

[0074] The substrate specificity of PtCCL1 and PtCCL2 is also differentfrom each other as determined using recombinant proteins produced in E.Coli. PtCCL1 utilized p-coumaric acid, caffeic acid, ferulic acid and5-hydroxyferulic acid as substrates. PtCCL2 showed activity top-coumaric acid, caffeic acid and ferulic acid but not to5-hydroxyferulic acid.

[0075] Specifically, PtCCl1 and PtCCL2 were used to construct expressionvectors for E.coli expression. The substrate specificity of PtCCL1 andPtCCL2 were tested using fusion proteins produced in E.coli. Twoplasmids, pQE/CCL1 and pQE/CCL2, were constructed in which the codingregions of PtCCL1 and PtCCL2, respectively were fused to N-terminal Histags in expression plasmids pQE-31 and pQE-32 (QIAGEN, Chatsworth,Calif.). The recombinant proteins of PtCCL1 and PtCCL2 produced byE.coli are approximately 59 kd and 63 kd, respectively.

[0076] The two recombinant proteins were tested for their activity inutilizing cinnamic acid derivatives. PtCCL1 recombinant protein showed100, 58, 71, 18 and 0% relative activity to p-coumaric acid, caffeicacid, ferulic acid, 5-hydroxyferulic acid and sinapic acid,respectively. PtCCL2 recombinant protein exhibited 100, 14, 27, 0 and 0%relative activity to p-coumaric acid, caffeic acid, ferulic acid,5-hydroxyferulic acid and sinapic acid, respectively. Neitherrecombinant protein showed detectable activity to sinapic acid.

[0077] The results of the tissue-specific expression pattern andsubstrate specificity suggests that in addition to the general functionof CCL, PtCCL1 apparently is more related to lignin synthesis in thexylem tissue and PtCCL2 apparently is more likely involved in flavonoidsynthesis and UV protection.

[0078] It should be noted that the isolation and characterization of thePtCCL1 and PtCCL2 cDNA clones is described in Kawaoka A, Chiang VL(1995), The Molecular Cloning and Expression of Syringyl- andGuaiacyl-Specific Hydroxycinnamate: CoA Ligases from Aspen (Populustremuloides ), Proceedings of the 6th International Conference onBiotechnology in the Pulp and Paper Industry, Vienna, Austria; and inHu, Wen-Jing, Isolation and Characterization of p-coumarate Co-enzyme Aligase cDNAs and Genes from Quaking Aspen (Populous temuloides Michx),Ph.D Dissertation, Michigan Technological University, Houghton, Mich.(1997); which are both herein incorporated by reference.

[0079] C. Transformation and Regeneration

[0080] Several methods for gene transformation of plant species with theCCL sequence are available such as the use of a transformation vector,agroinfection, electroinjection, particle bombardment with a gene gun ormicroinjection.

[0081] Preferably, a CCL cDNA clone is positioned in a binary expressionvector in an antisense orientation under the control of doublecauliflower mosiac virus 35S promoter. The vector is then preferablymobilized into a strain of Agrobacterium species such as tumefaciensstrain C58/pMP90 and is used as the DNA delivery system due to itsefficiency and low cost.

[0082] For example, with reference to FIG. 2, the binary expressionpACCL1 used for plant transformations is shown. Specifically, the PtCCL1cDNA is inserted in an antisense orientation into Pac I and BamH I sitesbetween the double CaMV 35S/AMV RNA4 and the 3′ terminator sequence ofthe nopaline synthase gene in a binary cloning vector pACCL1 (FIG. 2).The binary vector containing hygromycin phosphotransferase (HPT) gene ismodified from pBin 19.

[0083] The gene construct pACCL1 is available from MichiganTechnological University, Institute of Wood Research, Houghton, Mich.

[0084] The binary vector construct is mobilized in Agrobacteriumtumefaciens using the freeze-thaw method of Holsters et al., Mol. Gen.Genet. 163:181-187 (1978). For the freeze-thaw method, 1.5 ml ofovernight cultures Agrobacterium tumefaciens strain C58/pMP90 ispelleted at 5000×g for 3 minutes at 40 C and suspended in 1 ml of icecold 20 mM CaCl₂. To the suspension is added 10 1 l binary vector DNA(from an alkaline lysis minipreparation) and mixed by pipetting. Themicrocentrifuge tube is then frozen in liquid nitrogen for 5 minutes andthawed at 370 C for 5 minutes. After being cooled on ice, 1 ml of LB isadded and the mixture is incubated at 280 C for 2 hours with gentleshaking. 200 1 l of the cells is spread onto LB plates containinggentamycin and kanamycin and incubated at 280 C for 2 days. Coloniesgrown on the selection plates are randomly picked or miniprep andrestriction enzyme digestion analysis is used to verify the integration.

[0085] The resulting binary vector containing Agrobacterium strain isused to transform quaking aspen according to Tsai et al.,Agrobacterium-Mediated Transformation of Quaking Aspen (Populoustemuloides) and Regeneration of Transgenic Plants, Plant Cell Rep.14:94-97 as set forth below.

[0086] Explants of young leaves from cuttings of aspen are obtained bycutting leaf disks of approximately 7 mm square from the young leavesalong the midrib of the leaves. The explants are surface sterilized in20% commercial bleach for 10 minutes followed by rinsing 3 times withsterile double-distilled water.

[0087] All of the culture media used includes the basal medium of woodyplant medium (WPM) as described in Lloyd et al., Proc. Int. Plant Prop.Soc. 30:421-437 (1980) and supplemented with 2% sucrose. 650 mg/Lcalcium gluconate and 500 mg/L MES are added as pH buffers as describedin Tsai, Plant Cell Reports, 1994. All culture media is adjusted to pH5.5 prior to the addition of 0.075% Difco Bacto Agar and then autoclavedat 1210 C and 15 psi for 20 minutes. Filter sterilized antibiotics areadded to all culture media after autoclaving. All culture media aremaintained at 23±10 C in a growth chamber with 16 hour photoperiods(1601E×m⁻²×S⁻¹) except for callus induction (as will be described later)which is maintained in the dark.

[0088] The sterilized explants are then inoculated with the mobilizedvector with an overnight-grown agrobacterial suspension containing 20 μMacetosyringone. After cocultivation for 2 days, the explants are washedin 1 mg/ml claforan and ticarcillin for 2 hours with shaking to killAgrobacterium. The explants are blotted dry with sterile Whatman No. 1filter paper and transferred onto callus induction medium containing 50mg/L kanamycin and 300 mg/L claforan to induce and select transformedcallus. The callus induction medium is the basal medium with theaddition of 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid(2,4-D) at concentrations of 0.5 mg/L and 1 mg/L, respectively, toinduce callus.

[0089] The kanamycin-resistant explants are then subcultured on freshcallus induction media every two weeks. Callus formation occurs afterapproximately four weeks. Formed callus are separated from the explantand subcultured periodically for further proliferation.

[0090] When the callus clumps reach approximately 3 mm in diameter, thecallus clumps are transferred to shoot regeneration medium. The shootregeneration medium is the basal medium containing 50 mg/L kanamycin,0.5 mg/L thidiazuron (TDZ) as a plant growth regulator and cefotaxime at300 mg/L to kill Agrobacterium. Shoots were regenerated about 4 weeksafter callus is transferred to regeneration medium. As soon as theshoots are regenerated, they are immediately transferred to hormone-freeelongation medium containing 50 mg/L kanamycin and, whenever necessary,cefotaxime (300 mg/L), to promote elongation. Green and healthy shootselongated to 2-3 cm in length are excised and planted separately in ahormone-free rooting medium containing 50 mg/L kanamycin. The efficientuptake of kanamycin by shoots during their rooting stage provides themost effective selection for positive transformants. Transgenic plantsare then transplanted into soil medium of vermiculite:peatmoss:perliteat 1:1:1 and grown in the greenhouse.

[0091] The above described transformation and regeneration protocol isreadily adaptable to other tree species. Other published transformationand regeneration protocols for tree species include Danekar et al.,Bio/Technology 5:587-590 (1987); McGranahan et al., Bio/Technology6:800-804 (1988); McGranahan et al., Plant Cell Reports 8:512-616(1990); Chen, phD Thesis, North Carolina State University, Raleigh, N.C.(1991); Sullivan et al., Plant Cell Reports 12:303-306 (1993); Huang etal., In Vitro Cell Dev. Bio. 4:201-207 (1991); Wilde et al., PlantPhysiol. 98:114-120 (1992); Minocha et al., 1986 Proc. TAPPI Researchand Development Conference, TAPPI Press, Atlanta, pp. 89-91 (1986);Parsons et al., Bio/Technology 4:533-536 (1986); Fillatti et al., Mol.Gen. Genet 206:192-199 (1987); Pythoud et al., Bio/Technology5:1323-1327 (1987); De Block, Plant Physiol. 93:1110-1116 (1990);Brasileiro et al., Plant Mol. Bio 17:441-452 (1991); Brasileiro et al.,Transgenic Res. 1:133-141 (1992); Howe et al., Woody Plant Biotech.,Plenum Press, New York, pp.283-294 (1991); Klopfenstein et al., Can. J.For. Res. 21:1321-1328 (1991); Leple et al., Plant Cell Reports11:137-141 (1992); and Nilsson et al. Transgenic Res. 1:209-220 (1992).

[0092] D. Phenotype Changes

[0093] The results of the transformation can be confirmed withconventional PCR and Southern analysis. For example, transferring CCLcDNA in an antisense orientation down regulates CCL in the tree.Expression of the CCL has been found to be blocked up to 96 percent insome transgenic trees.

[0094] After acclimation, the transgenic aspen display an unusualphenotype, including big curly leaves, thick diameters, longerinternodes, more young leaves in the shoot tip and a red pigmentation inthe petioles extending into midvein leaves. Red coloration of thedeveloping secondary xylem tissues is observed after peeling of the barkin the transgenic plants.

[0095] E. Accelerated Growth

[0096] Down regulation of CCL alters growth of the transgenic trees. Forexample, transformation with an antisense CCL sequence accelerates thegrowth of the tree. Enhanced growth is markedly noticeable at all ages.In particular, the transgenic trees show enhanced growth in the form ofthicker stems and enlarged leaves as compared to control trees. Thesecharacteristics are retained in the vegetative propagules of thesetransgenic trees. Table 2 sets forth exemplary data with respect toseveral lines of transgenic quaking aspen grown in the greenhouse aftereight months. Volume represents the overall quantitative growth of thetree. TABLE 2 Growth Measurement for Control and Transgenic PlantsAVERAGE LENGTH HEIGHT DIAMETER VOLUME OF INTERNODE PLANT # (cm) (cm)*(cm³)* (cm) Control 1 247.7 1.08 75.6 2.6 Control 2 250.2 1.01 66.8 2.811-1 304.8 1.15 105.5 3.1 11-2 248.9 1.01 66.4 3.4 11-3 241.3 0.84 44.63.2 11-4 288.3 0.94 66.7 3.4 11-5 246.4 0.92 54.6 3.3 11-7 226.7 1.1375.7 3.4 11-8 289.6 1.16 102.0 3.3 11-9 287.0 1.76 232.6 4.3  11-10252.7 0.83 45.6 3.1  11-11 247.7 0.86 48.0 3.5 12-1 247.7 1.1 78.4 2.712-2 199.4 0.96 48.1 2.5 12-6 294.6 0.92 65.2 3.2 16-1 227.3 0.95 53.72.8 16-2 278.1 0.97 68.5 3.4 16-3 265.4 1.09 82.5 3.5 17-2 243.8 0.8950.5 2.6

[0097] The averages for height, diameter, volume and average lengthbetween internodes for the control plants are as follows: Height (cm)248.95 Diameter (cm) 1.045 Volume (cm³) 71.2 Ave. Length of 2.7Internodes (cm)

[0098] With respect to height alone, for those transgenic plants (11-1,11-4, 11-8, 11-9, 12-6, 16-2, 16-3) having a statistically larger heightthan the control plants, the average height was 286.83 cm as compared tothe control plant average height of 248.95 cm.

[0099] With respect to diameter alone, for those transgenic plants(11-1, 11-7, 11-8, 11-9) having a statistically larger diameter than thecontrol plants, the average diameter was 1.30 cm as compared to thecontrol plant average diameter of 1.045 cm.

[0100] With respect to volume alone, for those transgenic plants (11-1,11-8, 11-9, 12-1, 16-3) having a statistically larger volume than thecontrol plants, the average volume was 120.2 cm³ as compared to thecontrol plant average volume of 71.2 cm³.

[0101] With respect to average length of internodes alone, for thosetransgenic plants (11-1, 11-2, 11-3, 11-4, 11-5, 11-7, 11-8, 11-9,11-10, 12-6, 16-2, 16-3) having a statistically larger average length ofinternodes than the control plants, the average average length ofinternodes was 3.39 cm as compared to the control plant average averagelength of internodes of 2.70 cm.

[0102] As demonstrated in Table 2, while there are variations in growthamong the transgenic trees, the average length of the internodes for thetransgenic trees is consistently and significantly higher than that ofthe control plants. Variations in the growth of the transgenic trees isnormal and to be expected. Preferably, a transgenic tree with aparticular growth rate is selected and this tree is vegetativelypropagated to produce an unlimited number of clones that all exhibit theidentical growth rate.

[0103] F. Lignin

[0104] Down regulation of lignin pathway CCL results in production oftrees with reduced lignin content.

[0105] The following table shows the reduction of lignin content and CCLenzyme activity in several transgenic aspen which have been transformedwith an homologous antisense CCL sequence. TABLE 3 Characterization ofTransgenic Aspen Plants Harboring Antisense CCL Sequence Lignin Content% Based On CCL % CCL Enzyme Transgenic Wood % Lignin Enzyme ActivityPlant # Weight Reduction Activity* Reduction control 21.4 0.0 868 0 11-120.5 4.2 1171 −25 11-2 19.2 10.3 515 45 11-3 20.9 2.3 922 6 11-4 19.77.9 1032 −19 11-5 19.7 7.9 691 20 11-7 19.9 7.0 578 38 11-8 20.2 5.6 69420 11-9 20.4 4.7 806 14  11-10 19.4 9.3 455 51  11-11 20.4 4.7 726 2212-1 12.8 40.2 49 95 12-2 12.6 41.1 62 93 12-3 11.9 44.4 61 94 12-6 19.87.5 786 16 16-1 12.8 40.2 35 96 16-2 20.6 3.7 780 17 16-3 21.0 1.9 79515 17-1 20.5 4.2 855 9 17-2 21.4 0.0 925 1

[0106] Lignin content was determined according to Chiang and Funaoka(1990) Holzforschung 44:147-155. CCL enzyme activity was determinedaccording to Ranjeva et al. (1976), Biochimie 58:1255-1262.

[0107] The data in Table 3 demonstrates a correlation between downregulation of CCL and reduction in lignin content.

[0108] Transgenic trees with reduced lignin content have an alteredphenotype in that the stem is more elastic to the touch and the leavesare typically curlier.

[0109] It should also be noted that for those transgenic trees (12-1,12-2, 12-3 and 16-1) with the approximately 40% reduction in lignincontent and the corresponding approximately 95% reduction in CCL enzymelevels, all of those transgenic trees had a consistent deep redcoloration in the wood of the plant. Accordingly, the deep red color canbe used as an identifier of reduced lignin content.

[0110] Down regulation of lignin pathway CCL also results in productionof trees with an altered lignin structure. Based upon thioacidolysis(Rolando et al. (1992) Thioacidolysis, Methods in Lignin Chemistry,Springer-Verlag, Berlin, pp 334-349) of plants 12-3 and 16-1, coniferylalcohol and sinapyl alcohol lignin units are significantly reduced inthese two trees as compared to the control tree, as shown in thefollowing table. TABLE 4 Altered Lingin Structure Coniferyl AlcoholSinapyl Alcohol Plant # Units* Units* control 733 1700 12-3 283 592 16-1247 445

[0111] The alteration of the frequency of the structural units in ligninof these transgenic trees is evidence that the overall structure oflignin in these plants has been genetically altered.

[0112] G. Cellulose Content

[0113] Down regulation of lignin pathway CCL results in increasedcellulose content of the transgenic plants. Analysis of control andtransgenic aspen for carbohydrate content demonstrate a higher cellulosecontent in the transgenic trees than the control trees. Particularly,the transgenic trees that have over 40% lignin reduction have about10-15% higher cellulose content than the control. Data is set forth inthe following tables for trees that were transformed with homologous CCLin an antisense orientation: TABLE 5 Analysis of Carbohydrate Componentsin Transgenic and Control Aspen Galac- Plant # Glucan Arabinan tanRhamnan Xylan Mannan Control 44.23% 0.47% 0.79% 0.37% 17.19% 1.91% 11-249.05% 0.36% 1.05% 0.38% 15.34% 2.04% 11-9 45.95% 0.40% 0.80% 0.37%17.12% 1.83%  11-10 47.49% 0.43% 0.99% 0.40% 16.24% 2.35% 12-3 50.83%0.55% 1.24% 0.48% 17.25% 1.77% 16-1 48.14% 0.56% 1.07% 0.48% 19.14%1.58% 16-2 46.55% 0.34% 0.82% 0.37% 16.75% 2.31%

[0114] TABLE 6 Comparison of Lignin and Cellulose (glucan) Contents inTransgenic and Control Aspen Lignin Cellulose Content % Content % Plant# on wood % reduction on wood % increase Control 21.4 0 44.23 0 11-219.2 10.3 49.05 11.0 11-9 20.4 4.7 45.95 3.9  11-10 19.4 9.3 47.49 7.412-3 11.9 44.5 50.83 15.0 16-1 12.8 40.2 48.14 8.8 16-2 20.6 3.7 46.555.2

[0115] The procedure for carbohydrate analysis utilized is as follows.About 100 mg of powdery woody tissue with sizes that pass a 80-meshscreen was hydrolyzed with 1 mL of 72% (W/W) H2S04 for 1 hr at 300 C.Samples were then diluted to 4% (W/W) H2SO4 with distilled water, fucosewas added as an internal standard, and a secondary hydrolysis wasperformed for 1 hr at 1210 C. After secondary hydrolysis, the sugarcontents of the hydrolysates are determined by anion exchange highperformance liquid chromatography using pulsed amperometric detection.Sugar contents are expressed as % of the weight of the woody tissueused. The above procedures are similar to those in a publication by R CPettersen and V H Schwandt, 1991, J. Wood Chem & Technol. 11:495-501.

[0116] H. Increased Disease Resistance

[0117] Down regulation of lignin pathway CCL results in production oftrees with increased disease resistance, and in particular, withincreased fungal pathogen resistance.

[0118] In particular, greenhouse transgenic aspen plants showed adisease resistance to fungi such as those which induce leaf-blightdisease.

[0119] I. Promoters

[0120] Two distinct genes encoding CCL and their promoters were cloned.The promoter of PtCCL1 can drive gene expression specifically in xylemtissue and the promotor for PtCCL2 confers gene expression exclusivelyin the epidermal tissues. These promoters can be used to manipulate geneexpression to engineer traits of interest in specific tissues of targetplants. The significance of the promoters is the application of thexylem-specific promoter to direct the expression of any relevant genesspecifically in the xylem for engineering lignin content, ligninstructure, enhanced tree growth, cellulose content and other value-addedwood qualities, etc. The importance of the epidermis-specific promoteris its ability to drive the expression of any relevant genesspecifically in epidermal tissues for engineering disease-, UV light-,cold-, heat-, drought-, and other stress resistance traits in trees.

[0121] Specifically, the promoters of the PtCCL1 and PtCCL2 wereconventionally isolated as follows. An aspen genomic library wasscreened with PtCCL1 cDNA and PtCCL2 partial cDNA fragment to isolategenomic clones of PtCCL1 and PtCCL2. Eleven and seven positive genomicclones were identified for PtCCL1 and PtCC12 gene, respectively. Among11 positive clones for PtCCL1, PtCCL1g-4 contained a full length codingsequence and at least 2 kb 5′ flanking regions. The restriction map ofPtCCL1g-4 is set forth at FIG. 3.

[0122] With respect to PtCCL2, restriction map analysis was performedonλDNA of positive genomic clone PtCCL2g-11. The restriction map ofPtCCL2g-11 is set forth at FIG. 4.

[0123] Approximately a 2.3 kb 5′ flanking region of PtCCL1 was digestedfrom PtCCL1g-4 using Xba I and Sac I sites and cloned into pGEM7Z Xba Iand Sac I sites. The subcloned PtCCL1 promoter was named p7Z-4XS and therestriction map of P7Z-4XS is set forth at Tab 5. The 5′ unilateraldeletion of p7Z-4XS was generated for DNA sequencing by exonucleaseIII/S1 nuclease digestion using Erase-a-Base System (Promega, Madison,Wis.). The deletion series was sequenced using a primer on pGEM7Zvector.

[0124] A 1.6 kb Hind III and EcoR I fragment containing a 1.2 kb 5′flanking region of PtCCL2 and 0.4 kb coding region of PtCCL2g-11 weresubcloned in pBluescript II SK+ Hind III and EcoR I sites. Therestriction map of the resulting clone, pSK-11HE, was determined bydigesting the plasmid with several restriction enzymes, as in set forthat FIG. 6. In order to determine the sequence of the PtCCL2 promoter,pSK-11HE was further digested into small fragments according to therestriction map and subcloned into vectors with suitable cloning sites.The DNA sequence was determined using M13 universal primer and reverseprimer on the vector.

[0125] The DNA sequences of the two promoters was determined andanalyzed using qTaq cycle sequencing Kit (USB, Cleveland, Ohio), andGENETYX-MAC 7.3 sequence analysis software from Software DevelopmentCo., Ltd. The nucleotide sequence of promoter region of PtCCL1 is setforth as SEQ ID NO:5 and the nucleotide sequence of the promoter regionof PtCCL2 is set forth as SEQ ID NO:6. The promoter gene constructsPtCCL1p and PtCCL2p are available from Michigan TechnologicalUniversity, Institute of Wood Research, Houghton, Mich.

[0126] Tissue-specific expression can be achieved by conventionallyfusing the promoters of PtCCL1 or PtCCL2 to a gene of interest andtransferred to a plant species via Agrobacterium. For the sake ofexample, the promoters of PtCCL1 and PtCCL2 were fused to a GUS reportergene as detailed below. However, it should be noted that genes otherthan the GUS reporter gene can be fused to these promoters for tissuespecific expression.

[0127] In order to construct PtCCL1 promoter-GUS binary vector, a 1 Kbfragment covering 5′-flanking region and 117 bp coding region of PtCCL1was subcloned into pGEM7Z Sph I and EcoR I sites for constructingpromoter-GUS binary vector. In this 1 kb DNA fragment, it is found thatone Xho I site locates at 486 bases proximal to the translation startsite and the EcoR I site at 117 bases downstream the translation site.This 0.6 Kb fragment was subcloned into pGEM7Z Xho I and EcoR I sitesand used as a template in PCR amplification.

[0128] In order to construct a promoter-GUS transcriptional fusion, aBamH I site was introduced in front of the translation start site ofPtCCL1 by PCR. PCR amplification was performed using p7Z-4XE as thetemplate, M13 universal primer on pGEM7Z vector as 5′ end primer andPtCCL1p-1 primer containing a BamH I site at the end is complementary toa sequence upstream of the translation start site. The reaction wascarried out in 100 1 l reaction mix containing l×pfu reaction buffer,200 1 leach dNTPs, 100 1M each primer and 5 units of pfu. The PCRreaction mixture was denatured at 940 C for 5 minutes followed by 30cycles of 940 C (1 minute), 550 C (1 minute), 720 C (1 minute, 30seconds) and was ended with a 5 minute extension at 720 C.

[0129] The amplified 0.6 Kb fragment was cloned and sequenced to confirmthe sequence. The engineered 0.6 Kb fragment was ligated to p7Z-4SEwhich was digested with Xho I and BamH I. In order to incorporate a HindIII site in the 5′ end of PtCCL1 promoter, the 1 kb Sph I-BamH I PtCCL1promoter region was the cloned into pNoTA (5 prime→3 prime Inc.,Boulder, Colo.) Sph I and BamH I site. The 1 Kb PtCCL1 promoter was thenreleased from pNoTA vector with Hind III and BamH digestion andsubsequently transcriptionally fused to pBI101 Hind III and BamH I sitesin front of GUS. The resulting binary vector was named PtCCL1p-GUS andis set forth at FIG. 7.

[0130] In order to construct PtCCL2 promoter-GUS binary vector, pSK-11HEwas digested with Sph I and EcoR I to release 0.2 Kb Sph I and EcoR Ifragment. The 0.2 Kb fragment was cloned into pGEM7Z Sph I and EcoR Isites. A primer, PtCCL2p-3′ (5′-CATCGGATCCTGAGATGGAAGGGAGTTTCT-3′) wasdesigned to be complementary to a sequence upstream of the translationstart site of PtCCL2 and to incorporate BamH I site at the end.Amplification was performed using p7Z11SE as a template, M13 universalprimer as the 5′ end primer and PtCCL2p-3 as the 3′ end primer. A PCRreaction was carried out and the amplified PCR product was cloned andsequenced to check the fidelity of the PCR amplification. The 0.2 Kb SphI-BamH I DNA fragment with correct sequence was fused to pSK-11HElinearized with Sph I and BamH I. The resulting plasmid was namedpSK-11HB. The promoter of PtCCL2 was then excised from pSK-11HB withHind III and BamH I and ligated to PBI101 Hind III and BamH I site tomake PtCCL2p-GUS transcriptional fusion binary vector as shown in FIG.8.

[0131] The PtCCL1p-GUS and PtCCL2p-GUS constructs are then mobilizedinto Agrobacterium tumefaciens strain C58/pMP90 by freeze and thawmethod as explained previously.

[0132] Leaf disk transformation of tobacco with these two Agrobacteriumconstructs is conducted according to the method of Horsch R. B. (1988)Leaf Disk Transformation, Plant Molecular Biology Manual, A5:1-9.Histochemical GUS staining of promoter-GUS transgenic tobacco plantsdemonstrated that the PtCCL1 promoter restricted GUS expression in xylemtissue whereas PtCCL2 promoter regulated GUS expression in epidermalcells.

1 15 1 1915 DNA Populus tremuloides Michx. (aspen) CDS (83)...(1687) 1ccctcgcgaa actccgaaaa cagagagcac ctaaaactca ccatctctcc ctctgcatct 60ttagcccgca atggacgcca ca atg aat cca caa gaa ttc atc ttt cgc tca 112 MetAsn Pro Gln Glu Phe Ile Phe Arg Ser 1 5 10 aaa tta cca gac atc tac atcccg aaa aac ctt ccc ctg cat tca tac 160 Lys Leu Pro Asp Ile Tyr Ile ProLys Asn Leu Pro Leu His Ser Tyr 15 20 25 gtt ctt gag aac ttg tct aaa cattca tca aaa cct tgc ctg ata aat 208 Val Leu Glu Asn Leu Ser Lys His SerSer Lys Pro Cys Leu Ile Asn 30 35 40 ggc gcg aat gga gat gtc tac acc tatgct gat gtt gag ctc aca gca 256 Gly Ala Asn Gly Asp Val Tyr Thr Tyr AlaAsp Val Glu Leu Thr Ala 45 50 55 aga aga gtt gct tct ggt ctg aac aag attggt att caa caa ggt gac 304 Arg Arg Val Ala Ser Gly Leu Asn Lys Ile GlyIle Gln Gln Gly Asp 60 65 70 gtg atc atg ctc ttc cta cca agt tca cct gaattc gtg ctt gct ttc 352 Val Ile Met Leu Phe Leu Pro Ser Ser Pro Glu PheVal Leu Ala Phe 75 80 85 90 cta ggc gct tca cac aga ggt gcc atg atc actgct gcc aat cct ttc 400 Leu Gly Ala Ser His Arg Gly Ala Met Ile Thr AlaAla Asn Pro Phe 95 100 105 tcc acc cct gca gag cta gca aaa cat gcc aaggcc tcg aga gca aag 448 Ser Thr Pro Ala Glu Leu Ala Lys His Ala Lys AlaSer Arg Ala Lys 110 115 120 ctt ctg ata aca cag gct tgt tac tac gag aaggtt aaa gat ttt gcc 496 Leu Leu Ile Thr Gln Ala Cys Tyr Tyr Glu Lys ValLys Asp Phe Ala 125 130 135 cga gaa agt gat gtt aag gtc atg tgc gtg gactct gcc ccg gac ggt 544 Arg Glu Ser Asp Val Lys Val Met Cys Val Asp SerAla Pro Asp Gly 140 145 150 gct tca ctt ttc aga gct cac aca cag gca gacgaa aat gaa gtg cct 592 Ala Ser Leu Phe Arg Ala His Thr Gln Ala Asp GluAsn Glu Val Pro 155 160 165 170 cag gtc gac att agt cct gat gat gtc gtagca ttg cct tat tca tca 640 Gln Val Asp Ile Ser Pro Asp Asp Val Val AlaLeu Pro Tyr Ser Ser 175 180 185 ggg act aca ggg ttg cca aaa ggg gtc atgtta acg cac aaa ggg cta 688 Gly Thr Thr Gly Leu Pro Lys Gly Val Met LeuThr His Lys Gly Leu 190 195 200 ata acc agt gtg gct caa cag gta gat ggagac aat cct aac ctg tat 736 Ile Thr Ser Val Ala Gln Gln Val Asp Gly AspAsn Pro Asn Leu Tyr 205 210 215 ttt cac agt gaa gat gtg att ctg tgt gtgctt cct atg ttc cat atc 784 Phe His Ser Glu Asp Val Ile Leu Cys Val LeuPro Met Phe His Ile 220 225 230 tat gct ctg aat tca atg atg ctc tgt ggtctg aga gtt ggt gcc tcg 832 Tyr Ala Leu Asn Ser Met Met Leu Cys Gly LeuArg Val Gly Ala Ser 235 240 245 250 att ttg ata atg cca aag ttt gag attggt tct ttg ctg gga ttg att 880 Ile Leu Ile Met Pro Lys Phe Glu Ile GlySer Leu Leu Gly Leu Ile 255 260 265 gag aag tac aag gta tct ata gca ccagtt gtt cca cct gtg atg atg 928 Glu Lys Tyr Lys Val Ser Ile Ala Pro ValVal Pro Pro Val Met Met 270 275 280 gca att gct aag tca cct gat ctt gacaag cat gac ctg tct tct ttg 976 Ala Ile Ala Lys Ser Pro Asp Leu Asp LysHis Asp Leu Ser Ser Leu 285 290 295 agg atg ata aaa tct gga ggg gct ccattg ggc aag gaa ctt gaa gat 1024 Arg Met Ile Lys Ser Gly Gly Ala Pro LeuGly Lys Glu Leu Glu Asp 300 305 310 act gtc aga gct aag ttt cct cag gctaga ctt ggt cag gga tat gga 1072 Thr Val Arg Ala Lys Phe Pro Gln Ala ArgLeu Gly Gln Gly Tyr Gly 315 320 325 330 atg acc gag gca gga cct gtt ctagca atg tgc ttg gca ttt gcc aag 1120 Met Thr Glu Ala Gly Pro Val Leu AlaMet Cys Leu Ala Phe Ala Lys 335 340 345 gaa cca ttc gac ata aaa cca ggtgca tgt gga act gta gtc agg aat 1168 Glu Pro Phe Asp Ile Lys Pro Gly AlaCys Gly Thr Val Val Arg Asn 350 355 360 gca gag atg aag att gtt gac ccagaa aca ggg gtc tct cta ccg agg 1216 Ala Glu Met Lys Ile Val Asp Pro GluThr Gly Val Ser Leu Pro Arg 365 370 375 aac cag cct ggt gag atc tgc atccgg ggt gat cag atc atg aaa gga 1264 Asn Gln Pro Gly Glu Ile Cys Ile ArgGly Asp Gln Ile Met Lys Gly 380 385 390 tat ctt aat gac ccc gag gca acctca aga aca ata gac aaa gaa gga 1312 Tyr Leu Asn Asp Pro Glu Ala Thr SerArg Thr Ile Asp Lys Glu Gly 395 400 405 410 tgg ctg cac aca ggc gat atcggc tac att gat gat gat gat gag ctt 1360 Trp Leu His Thr Gly Asp Ile GlyTyr Ile Asp Asp Asp Asp Glu Leu 415 420 425 ttc atc gtt gac aga ttg aaggaa ttg atc aag tat aaa ggg ttt cag 1408 Phe Ile Val Asp Arg Leu Lys GluLeu Ile Lys Tyr Lys Gly Phe Gln 430 435 440 gtt gct cct act gaa ctc gaagct ttg tta ata gcc cat cca gag ata 1456 Val Ala Pro Thr Glu Leu Glu AlaLeu Leu Ile Ala His Pro Glu Ile 445 450 455 tcc gat gct gct gta gta ggattg aaa gat gag gat gcg gga gaa gtt 1504 Ser Asp Ala Ala Val Val Gly LeuLys Asp Glu Asp Ala Gly Glu Val 460 465 470 cct gtt gca ttt gta gtg aaatca gaa aag tct cag gcc acc gaa gat 1552 Pro Val Ala Phe Val Val Lys SerGlu Lys Ser Gln Ala Thr Glu Asp 475 480 485 490 gaa att aag cag tat atttca aaa cag gtg atc ttc tac aag aga ata 1600 Glu Ile Lys Gln Tyr Ile SerLys Gln Val Ile Phe Tyr Lys Arg Ile 495 500 505 aaa cga gtt ttc ttc attgaa gca att ccc aag gca cca tca ggc aag 1648 Lys Arg Val Phe Phe Ile GluAla Ile Pro Lys Ala Pro Ser Gly Lys 510 515 520 atc ctg agg aag aat ctgaaa gag aag ttg cca ggc ata taactgaaga 1697 Ile Leu Arg Lys Asn Leu LysGlu Lys Leu Pro Gly Ile 525 530 535 tgttactgaa catttaaccc tctgtcttatttctttaata cttgcgaatc attgtagtgt 1757 tgaaccaagc atgcttggaa aagacacgtacccaacgtaa gacagttact gttcctagta 1817 tacaagctct ttaatgttcg ttttgaacttgggaaaacat aagttctcct gtcgccatat 1877 ggagtaattc aattgaatat tttggtttctttaatgat 1915 2 535 PRT Populus tremuloides Michx. (aspen) 2 Met Asn ProGln Glu Phe Ile Phe Arg Ser Lys Leu Pro Asp Ile Tyr 1 5 10 15 Ile ProLys Asn Leu Pro Leu His Ser Tyr Val Leu Glu Asn Leu Ser 20 25 30 Lys HisSer Ser Lys Pro Cys Leu Ile Asn Gly Ala Asn Gly Asp Val 35 40 45 Tyr ThrTyr Ala Asp Val Glu Leu Thr Ala Arg Arg Val Ala Ser Gly 50 55 60 Leu AsnLys Ile Gly Ile Gln Gln Gly Asp Val Ile Met Leu Phe Leu 65 70 75 80 ProSer Ser Pro Glu Phe Val Leu Ala Phe Leu Gly Ala Ser His Arg 85 90 95 GlyAla Met Ile Thr Ala Ala Asn Pro Phe Ser Thr Pro Ala Glu Leu 100 105 110Ala Lys His Ala Lys Ala Ser Arg Ala Lys Leu Leu Ile Thr Gln Ala 115 120125 Cys Tyr Tyr Glu Lys Val Lys Asp Phe Ala Arg Glu Ser Asp Val Lys 130135 140 Val Met Cys Val Asp Ser Ala Pro Asp Gly Ala Ser Leu Phe Arg Ala145 150 155 160 His Thr Gln Ala Asp Glu Asn Glu Val Pro Gln Val Asp IleSer Pro 165 170 175 Asp Asp Val Val Ala Leu Pro Tyr Ser Ser Gly Thr ThrGly Leu Pro 180 185 190 Lys Gly Val Met Leu Thr His Lys Gly Leu Ile ThrSer Val Ala Gln 195 200 205 Gln Val Asp Gly Asp Asn Pro Asn Leu Tyr PheHis Ser Glu Asp Val 210 215 220 Ile Leu Cys Val Leu Pro Met Phe His IleTyr Ala Leu Asn Ser Met 225 230 235 240 Met Leu Cys Gly Leu Arg Val GlyAla Ser Ile Leu Ile Met Pro Lys 245 250 255 Phe Glu Ile Gly Ser Leu LeuGly Leu Ile Glu Lys Tyr Lys Val Ser 260 265 270 Ile Ala Pro Val Val ProPro Val Met Met Ala Ile Ala Lys Ser Pro 275 280 285 Asp Leu Asp Lys HisAsp Leu Ser Ser Leu Arg Met Ile Lys Ser Gly 290 295 300 Gly Ala Pro LeuGly Lys Glu Leu Glu Asp Thr Val Arg Ala Lys Phe 305 310 315 320 Pro GlnAla Arg Leu Gly Gln Gly Tyr Gly Met Thr Glu Ala Gly Pro 325 330 335 ValLeu Ala Met Cys Leu Ala Phe Ala Lys Glu Pro Phe Asp Ile Lys 340 345 350Pro Gly Ala Cys Gly Thr Val Val Arg Asn Ala Glu Met Lys Ile Val 355 360365 Asp Pro Glu Thr Gly Val Ser Leu Pro Arg Asn Gln Pro Gly Glu Ile 370375 380 Cys Ile Arg Gly Asp Gln Ile Met Lys Gly Tyr Leu Asn Asp Pro Glu385 390 395 400 Ala Thr Ser Arg Thr Ile Asp Lys Glu Gly Trp Leu His ThrGly Asp 405 410 415 Ile Gly Tyr Ile Asp Asp Asp Asp Glu Leu Phe Ile ValAsp Arg Leu 420 425 430 Lys Glu Leu Ile Lys Tyr Lys Gly Phe Gln Val AlaPro Thr Glu Leu 435 440 445 Glu Ala Leu Leu Ile Ala His Pro Glu Ile SerAsp Ala Ala Val Val 450 455 460 Gly Leu Lys Asp Glu Asp Ala Gly Glu ValPro Val Ala Phe Val Val 465 470 475 480 Lys Ser Glu Lys Ser Gln Ala ThrGlu Asp Glu Ile Lys Gln Tyr Ile 485 490 495 Ser Lys Gln Val Ile Phe TyrLys Arg Ile Lys Arg Val Phe Phe Ile 500 505 510 Glu Ala Ile Pro Lys AlaPro Ser Gly Lys Ile Leu Arg Lys Asn Leu 515 520 525 Lys Glu Lys Leu ProGly Ile 530 535 3 1710 DNA Populus tremuloides Michx. (aspen) CDS(1)...(1710) 3 atg atg tcc gtg gcc acg gtt gag ccc ccg aaa ccg gaa ctctcc cct 48 Met Met Ser Val Ala Thr Val Glu Pro Pro Lys Pro Glu Leu SerPro 1 5 10 15 cca caa aac caa aac gca cca tcc tct cat gaa act gat cacatt ttc 96 Pro Gln Asn Gln Asn Ala Pro Ser Ser His Glu Thr Asp His IlePhe 20 25 30 aga tca aaa cta cca gac ata acc atc tcg aac gac ctc cct ctgcac 144 Arg Ser Lys Leu Pro Asp Ile Thr Ile Ser Asn Asp Leu Pro Leu His35 40 45 gca tac tgc ttt gaa aac ctc tct gat ttc tca gat agg cca tgc ttg192 Ala Tyr Cys Phe Glu Asn Leu Ser Asp Phe Ser Asp Arg Pro Cys Leu 5055 60 att tca ggt tcc acg gga aaa acc tat tct ttt gcc gaa act cac ctc240 Ile Ser Gly Ser Thr Gly Lys Thr Tyr Ser Phe Ala Glu Thr His Leu 6570 75 80 ata tct cgg aag gtc gct gct ggg tta tcc aat ttg ggc atc aag aaa288 Ile Ser Arg Lys Val Ala Ala Gly Leu Ser Asn Leu Gly Ile Lys Lys 8590 95 ggc gat gta atc atg acc ctg ctc caa aac tgc cca gaa ttc gtc ttc336 Gly Asp Val Ile Met Thr Leu Leu Gln Asn Cys Pro Glu Phe Val Phe 100105 110 tcc ttc atc ggt gct tcc atg att ggt gca gtc atc acc act gcg aac384 Ser Phe Ile Gly Ala Ser Met Ile Gly Ala Val Ile Thr Thr Ala Asn 115120 125 cct ttc tac act caa agt gaa ata ttc aag caa ttc tct gct tct cgt432 Pro Phe Tyr Thr Gln Ser Glu Ile Phe Lys Gln Phe Ser Ala Ser Arg 130135 140 gcg aaa ctg att atc acc cag tct caa tat gtg aac aag cta gga gat480 Ala Lys Leu Ile Ile Thr Gln Ser Gln Tyr Val Asn Lys Leu Gly Asp 145150 155 160 agt gat tgc cat gaa aac aac caa aaa ccg ggg gaa gat ttc atagta 528 Ser Asp Cys His Glu Asn Asn Gln Lys Pro Gly Glu Asp Phe Ile Val165 170 175 atc acc att gat gac ccg cca gag aac tgt cta cat ttc aat gtgctt 576 Ile Thr Ile Asp Asp Pro Pro Glu Asn Cys Leu His Phe Asn Val Leu180 185 190 gtc gag gct agc gag agt gaa atg cca aca gtt tca atc ctt ccggat 624 Val Glu Ala Ser Glu Ser Glu Met Pro Thr Val Ser Ile Leu Pro Asp195 200 205 gat cct gtg gca tta cca ttc tct tca ggg aca aca ggg ctc ccaaaa 672 Asp Pro Val Ala Leu Pro Phe Ser Ser Gly Thr Thr Gly Leu Pro Lys210 215 220 gga gtg ata ctg acc cac aag agc ttg ata aca agt gtg gct caacaa 720 Gly Val Ile Leu Thr His Lys Ser Leu Ile Thr Ser Val Ala Gln Gln225 230 235 240 gtt gat gga gag atc cca aat tta tac ttg aaa caa gat gacgtt gtt 768 Val Asp Gly Glu Ile Pro Asn Leu Tyr Leu Lys Gln Asp Asp ValVal 245 250 255 tta tgc gtt tta cct ttg ttt cac atc ttt tca ttg aac agcgtg ttg 816 Leu Cys Val Leu Pro Leu Phe His Ile Phe Ser Leu Asn Ser ValLeu 260 265 270 tta tgc tcg ttg aga gcc ggt tct gct gtt ctt tta atg caaaag ttt 864 Leu Cys Ser Leu Arg Ala Gly Ser Ala Val Leu Leu Met Gln LysPhe 275 280 285 gag ata gga tca ctg cta gag ctc att cag aaa cac aat gtttcg gtt 912 Glu Ile Gly Ser Leu Leu Glu Leu Ile Gln Lys His Asn Val SerVal 290 295 300 gcg gct gtg gtg cca cca ctg gtg ctg gcg ttg gcc aag aaccca ttg 960 Ala Ala Val Val Pro Pro Leu Val Leu Ala Leu Ala Lys Asn ProLeu 305 310 315 320 gag gcg aac ttc gac ttg agt tcg atc agg gta gtc ctgtca ggg gct 1008 Glu Ala Asn Phe Asp Leu Ser Ser Ile Arg Val Val Leu SerGly Ala 325 330 335 gcg cca ctg ggg aag gag ctc gag gac gcc ctc agg agcagg gtt cct 1056 Ala Pro Leu Gly Lys Glu Leu Glu Asp Ala Leu Arg Ser ArgVal Pro 340 345 350 cag gcc atc ctg gga cag ggt tat ggg atg aca gag gccggg cct gtg 1104 Gln Ala Ile Leu Gly Gln Gly Tyr Gly Met Thr Glu Ala GlyPro Val 355 360 365 cta tca atg tgc tta gcc ttt tca aag caa cct ttc ccaacc aag tct 1152 Leu Ser Met Cys Leu Ala Phe Ser Lys Gln Pro Phe Pro ThrLys Ser 370 375 380 ggg tcg tgt gga acg gtg gtt aga aac gca gag ctc aaggtc att gac 1200 Gly Ser Cys Gly Thr Val Val Arg Asn Ala Glu Leu Lys ValIle Asp 385 390 395 400 cct gag acc ggt cgc tct ctt ggt tac aac caa cctggt gaa atc tgc 1248 Pro Glu Thr Gly Arg Ser Leu Gly Tyr Asn Gln Pro GlyGlu Ile Cys 405 410 415 atc cgt gga tcc caa atc atg aaa gga tat ttg aatgac gcg gaa gcc 1296 Ile Arg Gly Ser Gln Ile Met Lys Gly Tyr Leu Asn AspAla Glu Ala 420 425 430 acg gca aac acc ata gac gtt gag ggt tgg ctc cacact gga gat ata 1344 Thr Ala Asn Thr Ile Asp Val Glu Gly Trp Leu His ThrGly Asp Ile 435 440 445 ggt tat gtc gac gac gac gac gag att ttc att gttgat aga gtg aag 1392 Gly Tyr Val Asp Asp Asp Asp Glu Ile Phe Ile Val AspArg Val Lys 450 455 460 gaa atc ata aaa ttc aaa ggc ttc cag gtg ccg ccagcg gag ctt gag 1440 Glu Ile Ile Lys Phe Lys Gly Phe Gln Val Pro Pro AlaGlu Leu Glu 465 470 475 480 gct ctc ctt gta aac cac cct tca att gcg gatgcg gct gtt gtt ccg 1488 Ala Leu Leu Val Asn His Pro Ser Ile Ala Asp AlaAla Val Val Pro 485 490 495 caa aaa gac gag gtt gct ggt gaa gtt cct gtcgcg ttt gtg gtc cgc 1536 Gln Lys Asp Glu Val Ala Gly Glu Val Pro Val AlaPhe Val Val Arg 500 505 510 tca gat gat ctt gac ctt agt gaa gag gct gtaaaa gaa tac att gca 1584 Ser Asp Asp Leu Asp Leu Ser Glu Glu Ala Val LysGlu Tyr Ile Ala 515 520 525 aag cag gtg gtg ttc tac aag aaa ctg cac aaggtg ttc ttc gtt cat 1632 Lys Gln Val Val Phe Tyr Lys Lys Leu His Lys ValPhe Phe Val His 530 535 540 tct att ccc aaa tcg gct tct gga aag att ctaaga aaa gac ctc aga 1680 Ser Ile Pro Lys Ser Ala Ser Gly Lys Ile Leu ArgLys Asp Leu Arg 545 550 555 560 gcc aag ctt gcc aca gcc acc acc atg tcc1710 Ala Lys Leu Ala Thr Ala Thr Thr Met Ser 565 570 4 570 PRT Populustremuloides Michx. (aspen) 4 Met Met Ser Val Ala Thr Val Glu Pro Pro LysPro Glu Leu Ser Pro 1 5 10 15 Pro Gln Asn Gln Asn Ala Pro Ser Ser HisGlu Thr Asp His Ile Phe 20 25 30 Arg Ser Lys Leu Pro Asp Ile Thr Ile SerAsn Asp Leu Pro Leu His 35 40 45 Ala Tyr Cys Phe Glu Asn Leu Ser Asp PheSer Asp Arg Pro Cys Leu 50 55 60 Ile Ser Gly Ser Thr Gly Lys Thr Tyr SerPhe Ala Glu Thr His Leu 65 70 75 80 Ile Ser Arg Lys Val Ala Ala Gly LeuSer Asn Leu Gly Ile Lys Lys 85 90 95 Gly Asp Val Ile Met Thr Leu Leu GlnAsn Cys Pro Glu Phe Val Phe 100 105 110 Ser Phe Ile Gly Ala Ser Met IleGly Ala Val Ile Thr Thr Ala Asn 115 120 125 Pro Phe Tyr Thr Gln Ser GluIle Phe Lys Gln Phe Ser Ala Ser Arg 130 135 140 Ala Lys Leu Ile Ile ThrGln Ser Gln Tyr Val Asn Lys Leu Gly Asp 145 150 155 160 Ser Asp Cys HisGlu Asn Asn Gln Lys Pro Gly Glu Asp Phe Ile Val 165 170 175 Ile Thr IleAsp Asp Pro Pro Glu Asn Cys Leu His Phe Asn Val Leu 180 185 190 Val GluAla Ser Glu Ser Glu Met Pro Thr Val Ser Ile Leu Pro Asp 195 200 205 AspPro Val Ala Leu Pro Phe Ser Ser Gly Thr Thr Gly Leu Pro Lys 210 215 220Gly Val Ile Leu Thr His Lys Ser Leu Ile Thr Ser Val Ala Gln Gln 225 230235 240 Val Asp Gly Glu Ile Pro Asn Leu Tyr Leu Lys Gln Asp Asp Val Val245 250 255 Leu Cys Val Leu Pro Leu Phe His Ile Phe Ser Leu Asn Ser ValLeu 260 265 270 Leu Cys Ser Leu Arg Ala Gly Ser Ala Val Leu Leu Met GlnLys Phe 275 280 285 Glu Ile Gly Ser Leu Leu Glu Leu Ile Gln Lys His AsnVal Ser Val 290 295 300 Ala Ala Val Val Pro Pro Leu Val Leu Ala Leu AlaLys Asn Pro Leu 305 310 315 320 Glu Ala Asn Phe Asp Leu Ser Ser Ile ArgVal Val Leu Ser Gly Ala 325 330 335 Ala Pro Leu Gly Lys Glu Leu Glu AspAla Leu Arg Ser Arg Val Pro 340 345 350 Gln Ala Ile Leu Gly Gln Gly TyrGly Met Thr Glu Ala Gly Pro Val 355 360 365 Leu Ser Met Cys Leu Ala PheSer Lys Gln Pro Phe Pro Thr Lys Ser 370 375 380 Gly Ser Cys Gly Thr ValVal Arg Asn Ala Glu Leu Lys Val Ile Asp 385 390 395 400 Pro Glu Thr GlyArg Ser Leu Gly Tyr Asn Gln Pro Gly Glu Ile Cys 405 410 415 Ile Arg GlySer Gln Ile Met Lys Gly Tyr Leu Asn Asp Ala Glu Ala 420 425 430 Thr AlaAsn Thr Ile Asp Val Glu Gly Trp Leu His Thr Gly Asp Ile 435 440 445 GlyTyr Val Asp Asp Asp Asp Glu Ile Phe Ile Val Asp Arg Val Lys 450 455 460Glu Ile Ile Lys Phe Lys Gly Phe Gln Val Pro Pro Ala Glu Leu Glu 465 470475 480 Ala Leu Leu Val Asn His Pro Ser Ile Ala Asp Ala Ala Val Val Pro485 490 495 Gln Lys Asp Glu Val Ala Gly Glu Val Pro Val Ala Phe Val ValArg 500 505 510 Ser Asp Asp Leu Asp Leu Ser Glu Glu Ala Val Lys Glu TyrIle Ala 515 520 525 Lys Gln Val Val Phe Tyr Lys Lys Leu His Lys Val PhePhe Val His 530 535 540 Ser Ile Pro Lys Ser Ala Ser Gly Lys Ile Leu ArgLys Asp Leu Arg 545 550 555 560 Ala Lys Leu Ala Thr Ala Thr Thr Met Ser565 570 5 1172 DNA Populus tremuloides Michx. (aspen) 5 tgtaggattggtggaatggg atcattccta atcccttaat gacggtggca tgaacacaaa 60 gcaaagagaagttaggtcac tcctccttta tatatatata tatatgcatg catgaggacc 120 atggctatgatgaaggttaa tagaggtagt tgtgattgag atatgtccag cactagtttt 180 ttgttggtgtgatttctcat gatgacgcga aaattttata tatatatata atgaataata 240 tgattgattattctctgtaa ttttgtgaaa tagattaaaa cagctcaatg tgaggtgacc 300 agttgtcaaatgaccactcg acttggggca tggtgatttt tcaaatcaca actcaatttg 360 aaaactaaaattaaaaaaga tttagattat taaattatta ggttaattca cgggttggct 420 aatcaattattattaattaa aacgatagta tttttgataa tttaattaaa attttattgg 480 atttgaatgaactcaattac atcacaaaaa acctaatcaa attaatatct tatgtgatat 540 aatttagaaatataaatgat taacctttaa atctcgagtt tctcttataa aaaacacgta 600 taattgggctagatttaaca gctattattc aaactggcca ggacaattat taaaattaat 660 aattattattttttctaata aagcacttcc taattgttaa aatatatgtc taaacactaa 720 taataaaatttatttgtgta tctttggcag taggtgagag gtgctgacaa ataaattagt 780 gcataaaatataatggattg gtggtctgtg aaaagacagg tggaggacaa gccacctctc 840 tcaagtcaaaaggccatttc acaaccaacc caaatgggaa cccaccaccg ttccccgcca 900 ttaaaatccctaatctcacc aacccaactc cacagattct tcaccaaacg caactgattt 960 ttcaatcaatgttttcccta tactaccccc ccaacaactc cataataccc aatttgtcct 1020 ttcaccaacccccgtcctcc gtgccagcca attctatatc agcaggaatg ctctgcactc 1080 tgctttctcaggtctcctac cataagaaaa cagagagcac ctaaaactcg ccatctctcc 1140 ctctgcatctttagcccgca atggacgcga ca 1172 6 1180 DNA Populus tremuloides Michx.(aspen) 6 aagctttgag tattcatatg ggtattcatc cgaccattat ttttcaatttgtgttgtgtt 60 gatccaattt tcaacttatt tttttttcac ttatttttta ttagttatttttatttttat 120 tattttttta aaaatttaaa aattaaatta taacattttt attttatccctcattaacta 180 aaatagggat ggtaatagat attcatgaag ggagttatat atcaaatgatattagttaag 240 ctattttgat atttataccc tactcattac ttatggaata aaaaatttagatatttataa 300 aatatttatc ggatttcagg tattcatatg aatatttatt tgattattatttattcaaca 360 aaaaataaaa caattaatat gcatgtttga agtttatata tatattaagttaggtttaga 420 tagattttgg gtggggttaa ttaatattca taccctatct actatctatcaaataatcca 480 aataaattca cctaaattag gttgggtttg tattcatcaa gttaacattaaattgtaatt 540 ccgtaagtaa ctaaacaagt acaaagactt ctattttatc ttatatattaccataaagcc 600 aactatattt cctattcttt ttcatccctt ctatcgtaat tttctgtgacttttttattt 660 atatattaac ggtaacgaaa cacagcaata aaagttattg tgaaagatatggataattat 720 tatggtgact atgaaagagt aaatttgcca tgcactaagt tcctagtgtcatctcataaa 780 agacttgtct gccacgtaag ctgttgtgag tgtcgtttat ttacgcgtgtcaaccaatcg 840 ctgccaattg actcttgagg gtaggtgaga gcttcggctt tgatgggaactgcatgaggc 900 atagggtttg gtttcttgaa tgtgagatgg gcatgctttg gctcccttgctactcacctc 960 atcttcaatt tgccagctca gctaccagtc tctcaccact agtttcaccaaactttctct 1020 gctcctgtat ttattacacc ttgctcgatt ggctccgtcc tcgtacacgcatccacaccg 1080 atcgatcgat tagaaccata cagaattggg attggttggg tttacattctgcgttagata 1140 catctatcac agaaagaaac tcccttccat ctcaggaaac 1180 7 12PRT Populus tremuloides Michx. (aspen) 7 Leu Pro Tyr Ser Ser Gly Thr ThrGly Leu Pro Lys 1 5 10 8 7 PRT Populus tremuloides Michx. (aspen) 8 GlyGlu Ile Cys Ile Arg Gly 1 5 9 31 DNA Populus tremuloides Michx. (aspen)modified_base (11) n represents inosine 9 ttggatccgg nacnacnggnytnccnaarg g 31 10 28 DNA Populus tremuloides Michx. (aspen)modified_base (11) n represents inosine 10 ttggatccgt ngcncarcargtngaygg 28 11 27 DNA Populus tremuloides Michx. (aspen) modified_base(10) n represents inosine 11 atgtcgaccn ckdatrcada tytcncc 27 12 7 PRTPopulus tremuloides Michx. (aspen) unknown 12 Gly Glu Ile Cys Ile ArgGly 1 5 13 27 DNA Populus tremuloides Michx. (aspen) 13 tctgtctagatgatgtcgtg gccacgg 27 14 26 DNA Populus tremuloides Michx. (aspen) 14ttagatctct aggacatggt ggtggc 26 15 16 DNA Populus tremuloides Michx.(aspen) 15 cctttcacca accccc 16

We claim:
 1. A method for altering a characteristic of a tree comprisingthe step of incorporating into the genome of the tree a homologousnucleotide sequence encoding p-coumarate Co-enzyme A ligase in theantisense orientation such that when the nucleotide sequence isexpressed in the tree, the characteristic of the tree is altered,wherein the characteristic is selected from the group consisting ofaccelerated growth, reduced lignin content, altered lignin structure,increased disease resistance and increased cellulose content.
 2. Themethod as set forth in claim 1 wherein the nucleotide sequence isincorporated into the genome of the tree by transformation.
 3. Themethod as set forth in claim 2 wherein the transformation includes theuse of an Agrobacterium transfer vector.
 4. The method as set forth inclaim 1 wherein the nucleotide sequence is a cloned cDNA sequence ofp-coumarate Co-enzyme A ligase.
 5. The method as set forth in claim 1wherein the nucleotide sequence includes the gene promoter sequenceCaMV35S.
 6. The method as set forth in claim 1 wherein the tree is anangiosperm.
 7. The method as set forth in claim 1 wherein the tree is ofa forest tree species.
 8. The method as set forth in claim 1 wherein thealtered characteristic is accelerated growth and wherein the acceleratedgrowth is manifested as an increase in the average internode length. 9.The method as set forth in claim 1 wherein the altered characteristic isaccelerated growth and wherein the accelerated growth is manifested asan increase in tree height.
 10. The method as set forth in claim 1wherein the altered characteristic is accelerated growth and wherein theaccelerated growth is manifested as an increase in tree diameter. 11.The method as set forth in claim 1 wherein the altered characteristic isincreased disease resistance and wherein the increased diseaseresistance is increased fungal pathogen resistance.
 12. A tree having acharacteristic genetically altered through the incorporation into thegenome of the tree a homologous nucleotide sequence encoding p-coumarateCo-enzyme A ligase in the antisense orientation such that when thenucleotide sequence is expressed in the tree, the characteristic of thetree is altered, wherein the characteristic is selected from the groupconsisting of accelerated growth, reduced lignin content, altered ligninstructure, increased disease resistance and increased cellulose content.13. The tree as set forth in claim 12 wherein the nucleotide sequence isincorporated into the genome of the tree by transformation.
 14. The treeas set forth in claim 12 wherein the transformation includes the use ofan Agrobacterium transfer vector.
 15. The tree as set forth in claim 12wherein the nucleotide sequence is a cloned cDNA sequence of p-coumarateCo-enzyme A ligase.
 16. The tree as set forth in claim 12 wherein thenucleotide sequence includes the gene promoter sequence CaMV35S.
 17. Thetree as set forth in claim 12 wherein the tree is an angiosperm.
 18. Thetree as set forth in claim 12 wherein the tree is of a forest treespecies.
 19. The tree as set forth in claim 12 wherein the alteredcharacteristic is accelerated growth and wherein the accelerated growthis manifested as an increase in the average internode length.
 20. Thetree as set forth in claim 12 wherein the altered characteristic isaccelerated growth and wherein the accelerated growth is manifested asan increase in tree height.
 21. The tree as set forth in claim 12wherein the altered characteristic is accelerated growth and wherein theaccelerated growth is manifested as an increase in tree diameter. 22.The tree as set forth in claim 1 wherein the altered characteristic isincreased disease resistance and wherein the increased diseaseresistance is increased fungal pathogen resistance.
 23. A method foraltering a characteristic of a tree comprising the step of geneticallydown regulating the enzyme p-coumarate Co-enzyme A ligase, wherein thecharacteristic is selected from the group consisting of acceleratedgrowth, reduced lignin content, altered lignin structure, increaseddisease resistance and increased cellulose content.
 24. The method ofclaim 23 wherein the tree is genetically down regulated throughincorporation into the genome of the tree a homologous nucleotidesequence encoding p-coumarate Co-enzyme A ligase in the antisenseorientation.
 25. The method as set forth in claim 24 wherein thenucleotide sequence is incorporated into the genome of the tree bytransformation.
 26. The method as set forth in claim 25 wherein thetransformation includes the use of an Agrobacterium transfer vector. 27.The method as set forth in claim 24 wherein the nucleotide sequence is acloned cDNA sequence of p-coumarate Co-enzyme A ligase.
 28. The methodas set forth in claim 24 wherein the nucleotide sequence includes thegene promoter sequence CaMV35S.
 29. The method as set forth in claim 23wherein the tree is an angiosperm.
 30. The method as set forth in claim23 wherein the tree is of a forest tree species.
 31. A tree having acharacteristic altered by genetically down regulating the enzymep-coumarate Co-enzyme A ligase, wherein the characteristic is selectedfrom the group consisting of accelerated growth, reduced lignin content,altered lignin structure, increased disease resistance and increasedcellulose content.
 32. The tree of claim 31 wherein the tree isgenetically down regulated through incorporation into the genome of thetree a homologous nucleotide sequence encoding p-coumarate Co-enzyme Aligase in the antisense orientation.
 33. The tree of claim 32 whereinthe nucleotide sequence is incorporated into the genome of the tree bytransformation.
 34. The tree of claim 32 wherein the transformationincludes the use of an Agrobacterium transfer vector.
 35. The tree ofclaim 32 wherein the nucleotide sequence is a cloned cDNA sequence ofp-coumarate Co-enzyme A ligase.
 36. The tree of claim 32 wherein thenucleotide sequence includes the gene promoter sequence CaMV35S.
 37. Thetree of claim 31 wherein the tree is an angiosperm.
 38. The tree ofclaim 31 wherein the tree is of a forest tree species.
 39. Axylem-specific gene promoter that directs the expression of a gene inthe xylem of a tree.
 40. An epidermis-specific gene promoter thatdirects the expression of a gene in the epidermal tissue of a tree.